Nucleoside Tetra- and Pentaphosphates Prepared Using a Tetraphosphorylation Reagent Are Potent Inhibitors of Ribonuclease A The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Shepard, Scott M. et al. "Nucleoside Tetra- and Pentaphosphates Prepared Using a Tetraphosphorylation Reagent Are Potent Inhibitors of Ribonuclease A." Journal of the American Chemical Society 141, 46 (October 2019): 18400–18404 © 2019 American Chemical Society As Published http://dx.doi.org/10.1021/jacs.9b09760 Publisher American Chemical Society (ACS) Version Author's final manuscript Citable link https://hdl.handle.net/1721.1/128544 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author J Am Chem Manuscript Author Soc. Author Manuscript Author manuscript; available in PMC 2020 February 12. Published in final edited form as: J Am Chem Soc. 2019 November 20; 141(46): 18400–18404. doi:10.1021/jacs.9b09760. Nucleoside Tetra- and Pentaphosphates Prepared Using a Tetraphosphorylation Reagent Are Potent Inhibitors of Ribonuclease A Scott M. Shepard†, Ian W. Windsor†, Ronald T. Raines*, Christopher C. Cummins* Department of Chemistry, Massachusetts Institute of Technology, Cambridge Massachusetts 02139, United States Abstract Adenosine and uridine 5′-tetra- and 5′-pentaphosphates were synthesized from an activated tetrametaphosphate ([PPN]2[P4O11], [PPN]2[1], PPN = bis(triphenylphosphine)iminium) and subsequently tested for inhibition of the enzymatic activity of ribonuclease A (RNase A). Reagent [PPN]2[1] reacts with unprotected uridine and adenosine in the presence of a base under anhydrous conditions to give nucleoside tetrametaphosphates. Ring opening of these intermediates with tetrabutylammonium hydroxide ([TBA][OH]) yields adenosine and uridine tetraphosphates (p4A, p4U) in 92% and 85% yields, respectively, from the starting nucleoside. Treatment of ([PPN]2[1]) with AMP or UMP yields nucleoside-monophosphate tetrametaphosphates (cp4pA, cp4pU) having limited aqueous stability. Ring opening of these ultraphosphates with [TBA][OH] yields p5A and p5U in 58% and 70% yield from AMP and UMP, respectively. We characterized inorganic and nucleoside-conjugated linear and cyclic oligophosphates as competitive inhibitors of RNase A. Increasing the chain length in both linear and cyclic inorganic oligophosphates resulted in improved binding affinity. Increasing the length of oligophosphates on the 5′ position of adenosine beyond three had a deleterious effect on binding. Conversely, uridine nucleotides bearing 5′ oligophosphates saw progressive increases in binding with chain length. We solved X- ray cocrystal structures of the highest affinity binders from several classes. The terminal phosphate of p5A binds in the P1 enzymic subsite and forces the oligophosphate to adopt a convoluted conformation, while the oligophosphate of p5U binds in several extended conformations, targeting multiple cationic regions of the active-site cleft. Secretory ribonucleases (RNases) are a diverse family of enzymes that catalyze the cleavage of RNA to elicit biological functions ranging from cell signaling to innate immunity.1,2 *Corresponding Authors: [email protected]; [email protected]. †S.M.S. and I.W.W. contributed equally. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.9b09760. Synthetic details, spectra, kinetic data, and crystallographic data collection and refinement statistics (PDF) Accession Codes Structure data for the new compounds are available from the Protein Database under the following PDB codes: RNase A·cP6i complex, 6pvu; RNase A·p5A complex, 6pvv; RNase A·cp4pA complex, 6pvw; RNase A·p5U complex, 6pvx. The authors declare the following competing financial interest(s): The tetraphosphorylation reagent is covered in patent US10017388B2. Shepard et al. Page 2 Fundamental knowledge generated by studying RNase A, which derives from the bovine Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author pancreas, has shaped the fields of enzymology and protein chemistry.3,4 Furthermore, mammalian RNases have been shown to have angiogenic5 and neurotoxic activities,6 and targeted inhibitors of these enzymes may have human therapeutic potential.7 RNase A binds its substrates in enzymic subsites that interact with phosphoryl groups and nucleobases (Figure 1).8,9 Atypical nucleotides are among the best small-molecule inhibitors of RNase A. Diadenosine oligophosphates (Table 1, entries 5–7) are micromolar to submicromolar inhibitors that exhibit increasing affinity with longer phosphate chain lengths.10 Additionally, the highest affinity small-molecule inhibitors of RNase A, pyrophosphate-linked dinucleotides (Table 1, entries 1–4), have enhanced inhibition activity upon further phosphorylation.11 These observations prompted us to ask: can a simple oligophosphate on its own or appended to a single nucleoside serve as an effective small-molecule inhibitor of RNase A? In addition to canonical nucleoside mono-, di-, and triphosphates, nucleosides bearing longer oligophosphate chains are potent signaling molecules in biology.14–18 These and other related morphologies, such as dinucleotide oligophosphates,19–21 have been implicated in a variety of biological processes and ailments including hypertension22 and bacterial accumulation of polyphosphate.23–26 Nonetheless, the synthesis of oligophosphorylated compounds typically increases in difficulty with longer phosphate chains. Recently, methods have been developed to efficiently couple a triphosphate chain in one operation from 3− 14,27–32 trimetaphosphate (P3O9 ). Here, we extend this methodology to the 4− tetraphosphorylation of biomolecules, utilizing tetrametaphosphate (P4O12 ) to synthesize nucleoside tetraphosphates (p4N) and nucleoside pentaphosphates (p5N). Previous syntheses 33 of p4N have suffered from extremely low yields, requiring nucleoside triphosphates as starting materials,34 or iterative syntheses to add each additional phosphoryl group.13,23 The state-of-the-art synthesis of p4N involves coupling of trimetaphosphate and nucleoside 14 monophosphates. Here, we describe the facile synthesis of p4N and p5N by coupling tetrametaphosphate with nucleosides and nucleoside monophosphates, respectively (Figure 2). Utilizing tetraphosphorylation reagent 1 permits unprotected nucleosides to be converted to the corresponding p4N efficiently in a single operation. This is in contrast to the method of Taylor, which requires nucleoside monophosphates as the substrate.14 While enzymatic 35–37 methods have been reported, the few reported chemical syntheses of p5N have been limited in scope and low yielding.23,33 The activated tetrametaphosphate, [PPN]2[1], is synthesized by protonation of tetrametaphosphate and subsequent dehydration.38 Treatment of adenosine or uridine with [PPN]2[1] under rigorously anhydrous conditions leads to selective phosphorylation of the 5′ position. No satisfactory purification could be found for the resulting nucleoside- substituted tetrametaphosphates (cp4N, Figure 3), but treatment with [TBA][OH] results in ring opening to the linear tetraphosphates. HPLC purification in triethylammonium acetate buffer of the resulting mixtures gives adenosine tetraphosphate (p4A, Table 1, entry 18, 93% yield) and uridine tetraphosphate (p4U, Table 1, entry 25, 85% yield) as pure triethylammonium salts. J Am Chem Soc. Author manuscript; available in PMC 2020 February 12. Shepard et al. Page 3 Nucleoside 5′-pentaphosphates were obtained similarly by treatment of [PPN]2[1] with the Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author anhydrous TBA salts of pA and pU. The intermediate nucleoside-monophosphate substituted tetrametaphosphates, cp4pN, could be isolated in reasonable purity and were found to be stable in aqueous solution for several hours at room temperature before hydrolyzing to a mixture of nucleoside monophosphate, tetrametaphosphate, and nucleoside pentaphosphate. Treatment of cp4pN with excess [TBA][OH] results in selective ring opening to p5N in 24 h. The products were again purified by HPLC in triethylammonium acetate buffer, providing p5A (Table 1, entry 19, 58% yield) and p5U (Table 1, entry 26, 70% yield) as triethylammonium salts (Figure 3). Reagent 1 is highly moisture sensitive and must be prepared, stored, and utilized in an anhydrous environment, ideally inside a glovebox. We therefore developed a second phosphorylation methodology for the syntheses of both p4N and p5N, activating 38 [PPN]2[P4O12H2] in situ with dicyclohexylcarbodiimide (DCC) to form reagent 1. These reagents are bench stable, and this methodology can be utilized conveniently with a Schlenk line, although it suffers from lower yields (SI Sections 2.2 and 2.5). We performed inhibition kinetics using a fluorogenic substrate as described previously39 to assess the binding of oligophosphates to RNase A. In addition to the synthesized molecules, p4N and p5N, we assessed inhibition kinetics for a variety of inorganic phosphates to evaluate our hypothesis that longer oligophosphate chains increase binding affinity. Complementing previous reports of weak RNase A inhibition by orthophosphate (Pi, Table 1, entry 8) and pyrophosphate (P2i,